Residues in the Conserved Sequence (Gly-X-X-X-X-Gly-Lys-Thr/Ser) Are Located in the Domain Required for Stable Subunit-Subunit

The sequence (Gly-X-X-X-X-Gly-Lys-Thr/Ser) is conserved in nucleotide binding proteins including the a and ft subunits of the ATP synthase. Various mutations were introduced in the cLys-175 and aThr-176 residues in the sequence (Gly-Asp-Arg-Gln-Thr-Gly-Lys-Thr, residues 169-176) of the Escherichia coli ATP synthase a subunit. Surprisingly, single amino acid substitutions drastically affected the subunit assembly of the enzyme. The entire enzyme assembly was lost by aLys-175->Phe (or Trp) or aThr-176-»Phe (or Tyr) mutation. Other mutants had similar (aHis-175, cSer-175, aGly-175, aSer-176, and aHis-176 mutants) or lower (aAla-176, oCys-176, aLeu-176, and aVal-176 mutants) effects on assembly of the active enzyme compared with that of the wild-type. However, all these mutant enzymes except the aSer-176 enzyme showed enhanced cold sensitivities and reduced stabilities at high temperature. Mutant enzymes such as aGly-175 and orHis-176 showed low multi-site (steady state) catalysis, possibly due to loss of proper subunit-subunit interactions. These results suggest that the aLys-175 and aThr-176 residues are not absolutely essential for catalysis, but that they, or possibly the entire conserved sequence, are located in the key domain for the subunit-subunit interactions essential for enzym